Biogenesis of mitochondrial porin: The import pathway

Summary We review here the present knowledge about the pathway of import and assembly of porin into mitochondria and compare it to those of other mitochondrial proteins. Porin, like all outer mitochondrial membrane proteins studied so far is made as a precursor without a cleavble signal sequence; thus targeting information must reside in the mature sequence. At least part of this information appears to be located at the amino-terminal end of the molecule. Transport into mitochondria can occur post-translationally. In a first step, the porin precursor is specifically recognized on the mitochondrial surface by a protease sensitive receptor. In a second step, porin precursor inserts partially into the outer membrane. This step is mediated by a component of the import machinery common to the import pathways of precursor proteins destined for other mitochondrial subcompartments. Finally, porin is assembled to produce the functional oligomeric form of an integral membrane protein wich is characterized by its extreme protease resistance.     

Biogenesis of mitochondrial porin: the import pathway.

We review here the present knowledge about the pathway of import and assembly of porin into mitochondria and compare it to those of other mitochondrial proteins. Porin, like all outer mitochondrial membrane proteins studied so far is made as a precursor without a cleavable 'signal' sequence; thus targeting information must reside in the mature sequence. At least part of this information appears to be located at the amino-terminal end of the molecule. Transport into mitochondria can occur post-translationally. In a first step, the porin precursor is specifically recognized on the mitochondrial surface by a protease sensitive receptor. In a second step, porin precursor inserts partially into the outer membrane. This step is mediated by a component of the import machinery common to the import pathways of precursor proteins destined for other mitochondrial subcompartments. Finally, porin is assembled to produce the functional oligomeric form of an integral membrane protein which is characterized by its extreme protease resistance.     

Bacterial signal peptide recognizes HeLa cell mitochondrial import receptors and functions as a mitochondrial leader sequence

Phage display was used to identify new components of the mammalian mitochondrial receptor complex using Tom20 as a binding partner. Two peptides were identified. One had partial identity (SMLTVMA) with a bacterial signal peptide from Toho-1, a periplasmic protein. The other had partial identity with a mitochondrial inner membrane glutamate carrier. The bacterial signal peptide could carry a protein into mitochondria both in vivo and in vitro. The first six residues of the sequence, SMLTVM, were necessary for import but the two adjacent arginine residues in the 30-amino-acid leader were not critical for import. The signal peptides of Escherichia coli β-lactamase and Bacillsus subtilis lipase could not carry proteins into mitochondria. Presumably, the Toho-1 leader can adopt a structure compatible for recognition by the import apparatus.Received 29 April 2005; received after revision 8 June 2005; accepted 17 June 2005     

The cytosolic and membrane components required for peroxisomal protein import

Peroxisomes are vital intracellular organelles which house enzymes involved in a variety of metabolic pathways. The large number of human disorders associated with flawed peroxisome biogenesis emphasizes the importance of protein targeting to, and translocation across, the peroxisomal membrane. This brief review will summarize some of the emerging themes of peroxisomal protein import, specifically addressing the targeting signals possessed by constituent proteins, as well as the cytosolic, membrane and luminal components of the import machinery. Although a detailed understanding of the molecular mechanisms of peroxisomal protein import is not yet available, remarkable progress has been made in the field in recent years. An overview of these advances will be presented.     

The biogenesis and function of eukaryotic porins

Summary Like most other mitochondrial proteins porin is synthesized in the cytosol and imported posttranslationally into the outer mitochondrial membrane. This transport follows the general rules for mitochondrial, protein import with a few aberrations: a) porin contains an,uncleaved NH₂-terminal signal sequence, b) also its carboxyterminus might be involved in the import process, and c) this transport does not seem to require a membrane potential , although it is ATP-dependent. Most likely the actual import step occurs at contact sites between the outer and the inner mitochondrial membrane and involved at least one receptor protein.Although porin is known to be the major gate through the outer mitochondrial membrane, its absence only causes transient respiratory problems in yeast cells. This could mean a) that there is a bypass for some mitochondrial functions in the cytosol and/or b) that there are alternative channel proteins in the outer membrane. The first idea is supported by the overexpression of cytosolic virus-like particles in yeast cells lacking porin and the second by the occurrence of residual pore activity in mitochondrial outer membrane purified from porinless mutant cells.     

Degradation of the mitochondrial complex I assembly factor TMEM126B under chronic hypoxia

Cell stress such as hypoxia elicits adaptive responses, also on the level of mitochondria, and in part is mediated by the hypoxia-inducible factor (HIF) 1α. Adaptation of mitochondria towards acute hypoxic conditions is reasonably well understood, while regulatory mechanisms, especially of respiratory chain assembly factors, under chronic hypoxia remains elusive. One of these assembly factors is transmembrane protein 126B (TMEM126B). This protein is part of the mitochondrial complex I assembly machinery. We identified changes in complex I abundance under chronic hypoxia, in association with impaired substrate-specific mitochondrial respiration. Complexome profiling of isolated mitochondria of the human leukemia monocytic cell line THP-1 revealed HIF-1α-dependent deficits in complex I assembly and mitochondrial complex I assembly complex (MCIA) abundance. Of all mitochondrial MCIA members, we proved a selective HIF-1-dependent decrease of TMEM126B under chronic hypoxia. Mechanistically, HIF-1α induces the E3-ubiquitin ligase F-box/WD repeat-containing protein 1A (β-TrCP1), which in turn facilitates the proteolytic degradation of TMEM126B. Attenuating a functional complex I assembly appears critical for cellular adaptation towards chronic hypoxia and is linked to destruction of the mitochondrial assembly factor TMEM126B.     

Regulation of mitochondrial dynamics: convergences and divergences between yeast and vertebrates

In eukaryotic cells, the shape of mitochondria can be tuned to various physiological conditions by a balance of fusion and fission processes termed mitochondrial dynamics. Mitochondrial dynamics controls not only the morphology but also the function of mitochondria, and therefore is crucial in many aspects of a cell’s life. Consequently, dysfunction of mitochondrial dynamics has been implicated in a variety of human diseases including cancer. Several proteins important for mitochondrial fusion and fission have been discovered over the past decade. However, there is emerging evidence that there are as yet unidentified proteins important for these processes and that the fusion/fission machinery is not completely conserved between yeast and vertebrates. The recent characterization of several mammalian proteins important for the process that were not conserved in yeast, may indicate that the molecular mechanisms regulating and controlling the morphology and function of mitochondria are more elaborate and complex in vertebrates. This difference could possibly be a consequence of different needs in the different cell types of multicellular organisms. Here, we review recent advances in the field of mitochondrial dynamics. We highlight and discuss the mechanisms regulating recruitment of cytosolic Drp1 to the mitochondrial outer membrane by Fis1, Mff, and MIEF1 in mammals and the divergences in regulation of mitochondrial dynamics between yeast and vertebrates.     

Regulated protein degradation in mitochondria

Various adenosine triphosphate (ATP)-dependent proteases were identified within mitochondria which mediate selective mitochondrial protein degradation and fulfill crucial functions in mitochondrial biogenesis. The matrix-localized PIM1 protease, a homologue of theEscherichia coli Lon protease, is required for respiration and maintenance of mitochondrial genome integrity. Degradation of non-native polypeptides by PIM1 protease depends on the chaperone activity of the mitochondrial Hsp70 system, posing intriguing questions about the relation between the proteolytic system and the folding machinery in mitochondria. The mitochondrial inner membrane harbors two ATP-dependent metallopeptidases, them- and thei-AAA protease, which expose their catalytic sites to opposite membrane surfaces and cooperate in the degradation of inner membrane proteins. In addition to its proteolytic activity, them-AAA protease has chaperone-like activity during the assembly of respiratory and ATP-synthase complexes. It constitutes a quality control system in the inner membrane for membrane-embedded protein complexes.     

Evidence of undiscovered cell regulatory mechanisms: phosphoproteins and protein kinases in mitochondria

The finding that mitochondria contain substrates for protein kinases lead to the discovery that protein kinases are located in the mitochondria of certain tissues and species. These include pyruvate dyhydrogenase kinase, branched-chain α-ketoacid dehydrogenase kinase, protein kinase A, protein kinase Cδ, stress-activated kinase and A-Raf as well as unidentified kinases. Recent evidence suggests that mitochondrial protein kinases may be involved in physiological processes such as apoptosis and steroidogenesis. Additionally, the novel finding of low-molecular-weight GTP-binding proteins in mitochondria suggests the possibility that these may interact with mitochondrial protein kinases to regulate the activity of mitochondrial effector proteins. The fact that there are components of cellular regulatory systems in mitochondria indicates the exciting possibility of undiscovered systems regulating mitochondrial physiology. Received 19 June 2001; received after revision 7 August 2001; accepted 8 August 2001     

The biogenesis and function of eukaryotic porins.

Like most other mitochondrial proteins porin is synthesized in the cytosol and imported posttranslationally into the outer mitochondrial membrane. This transport follows the general rules for mitochondrial protein import with a few aberrations: a) porin contains an uncleaved NH2-terminal signal sequence, b) also its carboxyterminus might be involved in the import process, and c) this transport does not seem to require a membrane potential delta psi, although it is ATP-dependent. Most likely the actual import step occurs at contact sites between the outer and the inner mitochondrial membrane and involves at least one receptor protein. Although porin is known to be the major gate through the outer mitochondrial membrane, its absence only causes transient respiratory problems in yeast cells. This could mean a) that there is a bypass for some mitochondrial functions in the cytosol and/or b) that there are alternative channel proteins in the outer membrane. The first idea is supported by the overexpression of cytosolic virus-like particles in yeast cells lacking porin and the second by the occurrence of residual pore activity in mitochondrial outer membrane purified from porinless mutant cells.     

Megalin mediates plasma membrane to mitochondria cross-talk and regulates mitochondrial metabolism

Mitochondrial intracrines are extracellular signaling proteins, targeted to the mitochondria. The pathway for mitochondrial targeting of mitochondrial intracrines and actions in the mitochondria remains unknown. Megalin/LRP2 mediates the uptake of vitamins and proteins, and is critical for clearance of amyloid-β protein from the brain. Megalin mutations underlie the pathogenesis of Donnai–Barrow and Lowe syndromes, characterized by brain defects and kidney dysfunction; megalin was not previously known to reside in the mitochondria. Here, we show megalin is present in the mitochondria and associates with mitochondrial anti-oxidant proteins SIRT3 and stanniocalcin-1 (STC1). Megalin shuttles extracellularly-applied STC1, angiotensin II and TGF-β to the mitochondria through the retrograde early endosome-to-Golgi transport pathway and Rab32. Megalin knockout in cultured cells impairs glycolytic and respiratory capacities. Thus, megalin is critical for mitochondrial biology; mitochondrial intracrine signaling is a continuum of the retrograde early endosome-to-Golgi-Rab32 pathway and defects in this pathway may underlie disease processes in many systems.     

Hsp70 in mitochondrial biogenesis: From chaperoning nascent polypeptide chains to facilitation of protein degradation

The family of hsp70 (70 kilodalton heat shock protein) molecular chaperones plays an essential and diverse role in cellular physiology, Hsp70 proteins appear to elicit their effects by interacting with polypeptides that present domains which exhibit non-native conformations at distinct stages during their life in the cell. In this paper we review work pertaining to the functions of hsp70 proteins in chaperoning mitochondrial protein biogenesis. Hsp70 proteins function in protein synthesis, protein translocation across mitochondrial membranes, protein folding and finally the delivery of misfolded proteins to proteolytic enzymes in the mitochondrial matrix.     

Targeting mitochondria by α-tocopheryl succinate kills neuroblastoma cells irrespective of MycN oncogene expression

Amplification of the MycN oncogene characterizes a subset of highly aggressive neuroblastomas, the most common extracranial solid tumor of childhood. However, the significance of MycN amplification for tumor cell survival is controversial, since down-regulation of MycN was found to decrease markedly neuroblastoma sensitivity towards conventional anticancer drugs, cisplatin, and doxorubicin. Here, we show that a redox-silent analogue of vitamin E, α-tocopheryl succinate (α-TOS), which triggers apoptotic cell death via targeting mitochondria, can kill tumor cells irrespective of their MycN expression level. In cells overexpressing MycN, as well as cells in which MycN was switched off, α-TOS stimulated rapid entry of Ca(2+) into the cytosol, compromised Ca(2+) buffering capacity of the mitochondria and sensitized them towards mitochondrial permeability transition and subsequent apoptotic cell death. Prevention of mitochondrial Ca(2+) accumulation or chelation of cytosolic Ca(2+) rescued the cells. Thus, targeting mitochondria might be advantageous for the elimination of tumor cells with otherwise dormant apoptotic pathways.     

Mesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic glycolysis to the oxidative phosphorylation

We evaluated the energy metabolism of human mesenchymal stem cells (MSC) isolated from umbilical cord (UC) of preterm (< 37 weeks of gestational age) and term (≥ 37 weeks of gestational age) newborns, using MSC from adult bone marrow as control. A metabolic switch has been observed around the 34th week of gestational age from a prevalently anaerobic glycolysis to the oxidative phosphorylation. This metabolic change is associated with the organization of mitochondria reticulum: preterm MSCs presented a scarcely organized mitochondrial reticulum and low expression of proteins involved in the mitochondrial fission/fusion, compared to term MSCs. These changes seem governed by the expression of CLUH, a cytosolic messenger RNA-binding protein involved in the mitochondria biogenesis and distribution inside the cell; in fact, CLUH silencing in term MSC determined a metabolic fingerprint similar to that of preterm MSC. Our study discloses novel information on the production of energy and mitochondrial organization and function, during the passage from fetal to adult life, providing useful information for the management of preterm birth.     

Regulation of long-distance transport of mitochondria along microtubules

Mitochondria are cellular organelles of crucial importance, playing roles in cellular life and death. In certain cell types, such as neurons, mitochondria must travel long distances so as to meet metabolic demands of the cell. Mitochondrial movement is essentially microtubule (MT) based and is executed by two main motor proteins, Dynein and Kinesin. The organization of the cellular MT network and the identity of motors dictate mitochondrial transport. Tight coupling between MTs, motors, and the mitochondria is needed for the organelle precise localization. Two adaptor proteins are involved directly in mitochondria-motor coupling, namely Milton known also as TRAK, which is the motor adaptor, and Miro, which is the mitochondrial protein. Here, we discuss the active mitochondria transport process, as well as motor–mitochondria coupling in the context of MT organization in different cell types. We focus on mitochondrial trafficking in different cell types, specifically neurons, migrating cells, and polarized epithelial cells.     

Geranylgeranylacetone protects human monocytes from mitochondrial membrane depolarization independently of Hsp70 expression

The anti-ulcer drug geranylgeranylacetone (GGA) has been shown to induce the expression of heat shock proteins (HSPs), in particular of Hsp70, in gastric and small intestine cells. In this study, we investigated whether GGA was able to induce Hsp70 in another cell type, human monocytes, which represent a well-established model of Hsp70 expression under oxidative stress. In these cells, GGA had no significant effect either on basal or tobacco smoke-induced Hsp70 expression. We further investigated the effects of GGA on mitochondria, a key organelle of oxidant-mediated cell injury and a putative target for GGA-mediated protection. GGA significantly increased basal mitochondrial membrane polarization and inhibited the decrease in mitochondrial membrane potential of human monocytes exposed to distinct sources of clinically relevant oxidants such as tobacco smoke and y-irradiation. Our results indicate that mitochondria are targets for GGA-mediated protection against oxidative stress in human monocytes, independently of Hsp70.     

ATP-dependent proteases controlling mitochondrial function in the yeast Saccharomyces cerevisiae

Regulated protein degradation by ATP-dependent proteases plays a fundamental role in the biogenesis of mitochondria. Membrane-bound and soluble ATP-dependent proteases have been identified in various subcompartments of this organelle. Subunits composing these proteases are evolutionarily conserved from yeast to humans and, in support of an endosymbiotic origin of mitochondria, evolved from prokaryotic ancestors: the PIM1/Lon protease is active in the matrix of mitochondria, while the i-AAA protease and the m-AAA protease mediate the turnover of inner membrane proteins. Most of the knowledge concerning the biogenesis and the physiological role of ATP-dependent proteases comes from studies in the yeast Saccharomyces cerevisiae. Proteases were found to be required for mitochondrial stasis, for the maintenance of the morphology of the organelle and for mitochondrial genome integrity. ATP-dependent proteolysis is crucial for the expression of mitochondrially encoded subunits of respiratory chain complexes and for the assembly of these complexes. Hence, mitochondrial ATP-dependent proteases exert multiple roles which are essential for the maintenance of cellular respiratory competence.     

Morphofunctional changes in the mitochondrial subpopulations of conceptus tissues during the placentation process

To establish the role of mitochondrial subpopulations in the mitochondrial maturation process, we studied morphological and functional changes in the mitochondria of different mammalian conceptus tissues during the organogenic and the placentation processes. Mitochondrial subpopulations of three different conceptus tissues, embryo and visceral yolk sac placenta on gestational days 11, 12 and 13 and placenta on days 12 and 13, were examined morphologically by transmission electron microscopy. Cytochrome oxidase activity and protein levels were also measured in each mitochondrial subpopulation. The results indicate two different mitochondrial subpopulation profiles: a homogeneous one, which corresponds to immature mitochondria, and a heterogeneous one, which represents the mature mitochondria. The three tissues studied show different morphologic and metabolic patterns of mitochondrial maturation during the placentation process, rendering them suitable as experimental models to establish the p ossible relationship between mitochondrial maturation and the mitochondrial subpopulations. Received 5 August 2002; received after revision 23 September 2002; accepted 8 October 2002 RID="*" ID="*"Corresponding author.